Akt and Apoptosis
Apoptosis (programmed cell death) plays essential roles in embryonic development and pathogenesis of various diseases, such as degenerative neuronal diseases and cardiovascular diseases (MacLellan et al. 1998, Barinaga 1997a, Baringaga 1997b). Therefore, recent work has led to the identification of various pro- and anti-apoptotic gene products that are involved in the regulation or execution of programmed cell death. Expression of anti-apoptotic genes, such as Bcl2 or Bcl-x, inhibits apoptotic cell death induced by various stimuli. On the other hand, expression of pro-apoptotic genes, such as Bax or Bad, leads to programmed cell death (Aams et al. 1998). The execution of programmed cell death is mediated by caspase-1 related proteinases, including caspase-1, caspase-3, caspase-7, caspase-8 and caspase-9 etc (Thorneberry et al. 1998).
Recently, two intracellular signaling pathways involved in the regulation of cell survival/death have been studied. Activation of apoptotic stimulating kinase1 (ASK1) leads to apoptosis in various cell types (Ichijo et al. 1997), while a pathway involving phosphoinositide 3-kinase (PI3K) and Akt leads to cytoprotection. It has been demonstrated that the activity of ASK1 is induced by tumor necrosis factor-alpha (TNFa) treatment or Fas ligation (Ichijo et al. 1997, Chang et al. 1998). Overexpression of ASK1 dominant negative mutants inhibit apoptosis induced by TNFa or Fas ligation, indicating that ASK1 plays important roles during TNFa or Fas ligation-induced apoptotic cell death. The molecular mechanism by which ASK1 induces apoptosis is not clear. It has been shown that ectopic expression of ASK1 leads to activation of various stress-activated signaling pathways, such as the MKK4/JNK and MKK6/p38 pathways, which may mediate ASK1-induced apoptosis (Ichijo et al. 1997).
The PI3K/Akt pathway also appears important for regulating cell survival/cell death (Kulik et al. Franke et al 1997, Kauffmann-Zeh et al, Hemmings 1997. Dudek et al. 1997). Survival factors, such as platelet derived growth factor (PDGF), nerve growth factor (NGF) and insulin-like growth factor-1 (IGF-1), promote cell survival under various conditions by inducing the activity of PI3K (Kulik et al. 1997, Hemmings 1997). Activated PI3K leads to the production of phosphatidylinositol (3,4,5)-triphosphate (Ptdlns(3,4,5)-P3), which in turn binds to and induces the activity of a AH/PH-domain containing serine/threonine kinase, Akt (Franke et al 1995, Hemmings 1997b, Downward 1998, Alessi et al. 1996). Specific inhibitors of PI3K or dominant negative Akt mutants abolish survival-promoting activity of these growth factors or cytokines. In addition, introduction of constitutively active PI3K or Akt mutants promotes cell survival under conditions in which cells normally undergo apoptotic cell death (Kulik et al. 1997, Dudek et al. 1997). These observations demonstrate that the PI3K/Akt pathway plays important roles for regulating cell survival or apoptosis.
Two isoforms of human Akt protein kinases, Akt1 and Akt2 have been identified (Staal. 1987). A rat Akt sequence has also been identified (Konishi et al. 1995). Serine-473 in the C-terminus of human Akt1 has been shown to be critical for its regulation (Stokeo et al. 1997; Stephens et al. 1998). Upon growth factor stimulation, PI3K is activated. The product of PI3K, Ptdlns(3.4.5)-P binds Akt1, and causes translocation of Akt1 from the cytoplasm to the proximity of the inner cytoplasmic membrane, where it becomes phosphorylated at residues Thr308 and Ser473 (Downward, 1998). Phosphorylation of these residues is critical for the activation of Akt1. A recently identified protein kinase, PDK1, has been shown to be responsible for the phosphorylation of Thr308, while the kinase(s) which phosphorylates Ser473 has not yet been identified (Stokeo et al. 1997, Stephens et al. 1998).
Gene Therapy
Gene therapy involves correcting a deficiency or abnormality (mutation, aberrant expression, and the like) by introduction of genetic information into a patient, such as into an affected cell or organ of the patient. This genetic information may be introduced either in vitro into a cell, the modified cell then being reintroduced into the body, or directly in vivo into an appropriate site. In this connection, different techniques of transfection and of gene transfer have been described in the literature (see Roemer and Friedman, Eur. J. Biochem. 208 (1992) 211), including transfection of “naked DNA” and various techniques involving complexes of DNA and DEAE-dextran (Pagano et al., J. Virol. 1 (1967) 891), of DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375), of DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), the use of liposomes (Fraley et al., J. Biol. Chem. 255 (1980) 10431) and the like. More recently, the use of viruses as vectors for the transfer of genes has emerged as a promising alternative to physical transfection techniques. In this regard, different viruses have been tested for their capacity to infect certain cell populations, including retroviruses, herpes viruses, adeno-associated viruses, and adenoviruses.
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